Lactic Acid or 2-Hydroxypropionic Acid has wide applications in diverse areas like food, pharmaceuticals, cosmetics, green solvents, specialty chemicals, textile, leather and biodegradable plastics/polymers. Other potential applications include biocompatible polylactic acids for biomedical applications.
On account of its eco-friendliness, easy recyclability and cost effectiveness, the demand for lactic acid based bio-polymers has increased, thereby increasing the lactic acid consumption. It has been projected that the global demand for lactic acid would reach 328.9 thousand metric tons by year 2015.
At present, fermentation of sugars by lactic acid bacteria supplies all the optically pure lactic acid produced world-wide. However, industrial lactic acid production by fermentation starts with glucose derived from starch or sucrose, derived from food-based materials. One of the most critical and lucrative areas of interest at present is the production of L(+)-lactic acid from non edible and cheaper sources such as lignocellulosic biomass.
Several reports are available wherein the lactic acid is generated by fermentation of renewable agricultural feedstock resources such as corn, whey, potatoes, cane sugar, molasses of beet sugar, xylose stream generated from lignocellulosic biomass etc.
U.S. Pat. No. 4,963,486 issued to Hang, on Oct. 16, 1990 claims the production of L (+) lactic acid from Rhizopus oryzae as a single step fermentation process from renewable biomass namely corn, cassava, rice, oat, wheat, barley and sorghum representing starchy biomass. The patent claims that the fungus mentioned in the invention is capable of supplying all the required enzymes for both saccharification of gelatinised starch and fermentation to L (+)-lactic acid. Hang also discloses the production of 350 g of L (+)-lactic acid from one kg crushed corn.
Similarly Tsai et al in their U.S. Pat. No. 5,464,760 (7 Nov. 1995) claim the production of lactic acid using a consortium of Lactobacillus strains from Starch under SSF conditions in combination with α-amylase enzyme and thereafter its recovery in pure form from Sodium lactate using various methods like electro-dialysis. The invention of U.S. Pat. No. 5,464,760 involves the bioconversion of industrial food waste, such as potato waste, corn, rice, cheese whey, cane sugars, beet sugars or the like, containing starch to lactic acid suitable for conversion to photodegradable or biodegradable plastics.
The European Patent Application filed by Shimadzu Corporation (EP0770684A2) with a priority date of 27 Oct. 1995, claims the production of L-Lactic Acid with a purity of more than 70% to as high as 95% by Bacillus species in particular under anaerobic conditions mainly from glucose, sucrose, maltose, fructose, mannitol, lactose and Starch.
Numerous papers and patents have reported the production of L(+)-lactic acid through simultaneous saccharification and fermentation (SSF) route. However one of the major disadvantage quoted by Hofvendahl and Hahn-Hagerdal (Enzyme Microb. Technol., 2000; 26: 87-107) with the SSF process is the difference in the optimal conditions for enzymatic hydrolysis (pH<5.0 and temperature: 50° C.) and lactic acid fermentation (pH-5.0-7.0 and temperature being 37-43° C.).
Shin-ichiro Abe and Motoyoshi Takagi have shown the production of lactic acid using a combination of Trichoderma reesei as a source of cellulase enzyme for saccharification and Lactobacillus delbrueckii as the lactic acid producing microorganism. At the end of 120 hours 52.5 g/L of lactic acid was produced with only 6.2 g/L of reducing sugar left with an initial feed of 100 g/L of cellulose powder Type C (Biotechnol. Bioengg; 1991, 37: 93-96).
R. P. John et al (Braz. Arch. Biol. Technol.; 2008, 51 (6): 1241-1248) have also reported the production of L-lactic acid from cassava starch through SSF route using combination of L. delbrueckii and L. casei strain together with use of α-amylase and glucoamylase enzyme.
Mark S. Ou et al (J Ind Microbiol Biotechnol; 2011, 38:599-605) have also reported to produce L(+) Lactic Acid with 80% yield under fed batch SSF conditions of crystalline Cellulose with fungal enzymes dosed at 15 FPU/g Cellulose and Bacillus coagulans at pH-5 and temperature being 50° C.
In yet another patent application, Otto has claimed (US 2004/0203122 A1 and WO 2004/063382A2) the preparation of Lactic Acid through homolactic fermentation by a moderate thermophile of Bacillus under anaerobic conditions from glucose, xylose and arabinose derived from Biomass and grown in chemically defined medium.
Van Walsum et al in their Patent Application US 2011/0183389 have claimed the conversion of xylose to Lactic Acid from woody biomass which is prehydrolysed by acid or enzyme to generate an aqueous extract comprising of glucose, mannose, galactose, xylose and arabinose using Bacillus coagulans strain.
Very recently Direvo Industrial Biotechnology in their patent application WO 2013050584 A1 published on 11 Apr. 2013 has claimed the bioconversion of lignocellulosic biomass to Lactic acid. However, as per their claims the pretreatment step not only involves mechanical disprution using ball milling, but also physical pretreatment that involves use of steam, sulphuric acid, alkali but also biochemical step involving use of cellulose and hemicellulose degrading enzymes. The claimed thermophilic and xylanolytic bacteria Thermoanaerobacter is able to ferment the hydrolyzed lignocellulosic biomass under obligate anaerobic conditions with major product as L-lactic acid and acetic acid as by-product.
In the recent past, the conversion of cellobiose, to lactic acid has gained lot of importance. Adsul et al (AEM, August 2007, p. 5055-5057) have reported the production of 90 g/L L(+) Lactic Acid from 100 g/L of Cellobiose from a mutant strain of Lactobacillus delbrueckii showing aryl-β-glucosidase activity from whole cells, thereby suggesting that the enzyme is cell bound. This strain is known to utilize even cellotriose also efficiently.
Recently Mohamed Ali Abdel-Rahman et at (Appl Microbiol Biotechnol (2011) 89:1039-1049) have reported the production of optically pure L (+) Lactic Acid (˜35 g/L) when fed with cellobiose and glucose (20 g/L each) simultaneously at the end of 15 hours from Enterococcus mundtii QU 25 grown at 43° C. at pH-7.0.
Pratibha Dheeran et al (J Ind Microbiol Biotechnol; DOI 10.1007/s10295-012-1093-1) have described the xylanolytic activity of Paenibacillus macerans IIPSP3 (MTCC 5569) obtained from termite gut and its growth of various carbon sources, such as birchwood xylan, beechwood xylan, oatspelt xylan, carboxymethyl cellulose, cellobiose, glucose, xylose, and raw substrates, such as bagasse (untreated and pre-treated with 0.1% H2SO4 at 121° C. for 30 min), and corn cob chips (collected from nearby farms), at concentrations ranging from 0.1 to 2.5% (w/v). However they fail to report the lactic acid production from the isolated strain.
Thus none of the papers and the patents in the state of art, taken in combination or singly describes the process for a direct conversion of lignocellulosic biomass to L (+)-lactic Acid by thermophilic Paenibacillus macerans without any addition of external enzyme such as amylases or cellulases or their combination, that too under aerobic conditions.
Thus there is a need in the art for thermo-tolerant organisms capable of not only hydrolysing the cellulose rich lignocellulosic biomass, but also efficiently producing value added product such as L-lactic acid in a single step process. The present invention meets all these needs.
The present invention offers consolidated bio-processing of lignocellulosic biomass to L-Lactic Acid wherein the novel thermophilic strain of Paenibacillus IIPSP3 not only attacks the glucan and xylan, the principal constituents of biomass and breaks them of monomeric sugars but is also capable of fermenting these sugars to L-Lactic Acid under aerobic conditions. The cellulolytic activity of Paenibacillus macerans IIPSP3 (MTCC 5569) has been proven by fermentation of pure cellulosic substrates such as sodium salt of carboxy methyl cellulose, micro-crystalline cellulose, Avicel PH 101 and cellobiose to L-Lactic Acid.